Growing evidence suggests that AMPA/kainate (AMP/KA) as well as NMDA receptors participate in the pathogenesis of cerebral hypoxia-ischemia. Although links between NMDA receptor overactivation and hypoxic neuronal death are extensively studied, cellular mechanisms of AMPA/KA-receptor mediated hypoxic neuronal injury remain to be established. Project III examines the hypothesis that AMPA/KA receptor overactivation contributes to hypoxic neuronal injury by disrupting intracellular calcium homeostasis. The experiments in this project will focus on measurements of intracellular calcium ([Ca2+]i) in cultured cortical neurons using digital fluorescence and confocal microscopy with the cytosolic calcium indicators, fura-2 and indo-1. Alterations in calcium homeostasis will be compared with the experimental endpoint of neuronal survival. To isolate the contribution of AMPA/KA receptors, cultured neurons will be exposed to prolonged oxygen-glucose deprivation in the presence of NMDA receptor antagonists; previous experiments have established that the resulting neuronal injury is mediated largely by AMPA/KA receptor activation. The specific aims of this project are (1) to characterize the temporal profile and subcellular distribution of AMPA/KA receptor- mediated calcium entry during oxygen-glucose deprivation, (2) to examine the pharmacology of AMPA/KA receptor agonists and antagonists, including agents which alter receptor desensitization, and (3) to determine the routes of calcium entry triggered by AMPA/KA receptor overactivation.